Heart failure (HF) continues to be a leading cause of morbidity and mortality. A universal characteristic of human and experimental heart failure is impaired sarcoplasmic reticulum (SR) Ca-cycling, reflecting partially the depressed Ca-sequestration through SERCA2a/phospholamban (PLN). Despite recent advances, the mechanisms responsible for the deteriorated function caused by SERCA2a/PLN activity remain elusive. Importantly, our original simple understanding of PLN serving as the sole physiological brake to inhibit SERCA2a and contractility has recently evolved, as we have identified additional interacting proteins in this complex. These include the cytosolic anti-apoptotic HS-1 associated protein X-1 (HAX-1), the heat shock protein 90 and the intraluminal histidine-rich Ca-binding (HRC) protein. HAX-1 directly interacts with PLN and promotes PLN inhibition of SERCA2a, while Hsp90 binds to HAX-1 and provides an additional layer of PLN regulation, decreasing SERCA's Ca-affinity and contractility. In addition, HRC interacts with SERCA2a in the lumen of the SR and modulates the maximal rates of SR Ca-transport. HRC also interacts with triadin and regulates ryanodine receptor (RyR) activity. Thus, HRC may mediate an important link between SR Ca- uptake and Ca-release, impacting overall cardiomyocyte Ca-handling. Indeed, a human genetic variant in HRC (S96A) is associated with impaired SR Ca-cycling and arrhythmias in dilated cardiomyopathy patients. The central hypothesis of the proposed studies is that there is a fine cross-talk between Ca-handling and apoptotic proteins at the SR level which regulates contractility, survival and remodeling in the heart. The innovation of this proposal is the first identification of an Hsp90/HAX-1/PLN/SERCA2a/HRC/Triadin/RyR interactome, suggesting that perturbations in the levels or activity of a single protein in this network will influence the rest of the partners, eliciting amplification of its effects. Our goal i to define the triggers and mechanisms, which disrupt the function of this SR regulatory complex in response to clinically relevant stress and potentially reveal new therapeutic targets. Accordingly, our Aims will provide a first comprehensive characterization of the: a) functional significance of Hsp90/HAX-1 in regulation of PLN/SERCA2a activity and SR Ca-load, impacting cardiomyocyte contractility and apoptosis through the ER and mitochondria pathways, which may lead to remodeling under stress conditions; and b) the mechanisms underlying aberrant SR Ca- cycling by alterations in HRC levels or activity and their role in cardiomyocyte function and survival. We will employ an integrative approach with studies at the molecular, biochemical and physiological levels. Our pilot studies are exciting and suggest that the proposed experiments are feasible and highly relevant since the levels of HAX-1 and HRC decrease in failing hearts. The findings will provide new mechanistic insights underlying the role of impaired SR Ca-cycling in heart failure and arrhythmia development and may reveal new molecular pathways and targets associated with cardiomyocyte stress conditions.